Abstract 2472: β1 integrin-mediated repair of double-strand breaks in the mouse mammary gland

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL β 1 integrin signaling has been implicated in the progression and metastasis of various cancers and has been shown to facilitate resistance to radiation therapy. Our laboratory showed that extracellular matrix (ECM) signaling via β1 integrin downregulates homologous recombination (HR), as well as ionizing radiation (IR) induced foci formation of γ-H2AX, RAD51, and MRE11 in single non-dividing cells of a non-tumorigenic human breast epithelial cell line. When the cells were allowed to form junctions, ECM had the opposite effect on repair. Mouse mammary epithelial cells in primary culture behaved as junctioned human breast epithelial cells, upregulating HR and IR induced γ-H2AX foci formation. These results suggested the importance of the tissue microenvironment including cell-cell junctions. Here we hypothesized that β1 integrin-mediated repair depends on the context including homotypic and heterotypic cellular interactions. To test this hypothesis, we studied the effect of β1 integrin in the mouse mammary gland in vivo on DNA repair relevant outcomes. For determining the β1 integrin effect on foci kinetics, mice were intraperitonially injected with the function-blocking antibody Ha2/5 or control IgG. 24 hrs later, the mice were subjected to 6 Gy IR and the level of γ-H2AX foci formation determined by counting Z-stacks of confocal microscopy images. We found that blocking the β1 integrin receptor significantly decreased γ-H2AX foci formation at 15 min, 1 hr, and 2 hrs post IR. These time points correlated with both the formation and disappearance kinetics of the foci. Both the β1 blocked and control mammary glands resolved all foci successfully by 4 hrs, suggesting that overall repair was not attenuated by systemic β1 integrin blocking. We have also ablated the ITGB1 gene using the Cre/loxP recombination system. Cre endonuclease was delivered by up-the-teat injections into 8-12 week old pre-pubertal mice, epithelial organoids were isolated, β1 integrin and Cre endonuclease expression are determined by immunocytochemistry. Cre recombinase was successfully delivered to the ductal epithelial cells by up-the-teat viral injection, and resulted in reduced β1 integrin expression in ∼80% of the organoids isolated from injected mice. In addition MMTV/Cre and ITGB1 floxed mice were crossed to successfully excise the β1 integrin gene and reduce protein expression in the mammary glands of mice aged 10-13 weeks. Experiments are in progress to determine the effects of local and complete β1 integrin gene ablation on IR induced repair foci kinetics. These observations provide the proof-of-principle that ECM has a normal function in maintaining DNA repair processes via β1 integrin signaling in the complex in vivo microenvironment of the mouse mammary gland as well as in cultured cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2472. doi:10.1158/1538-7445.AM2011-2472